1. Field of the Invention
The present invention is broadly concerned with oligonucleotide-carbohydrate conjugates which are resistant to nuclease degradation and can be used as inhibitors of gene expression. More particularly, the invention pertains to such conjugates which are preferably formed using carbohydrates (e.g., sucrose) coupled via crosslinkers (e.g., 1,5-bis(succinimidooxycarbonyloxy)pentane) to the 3xe2x80x2-ends of oligonucleotides. The invention also relates to a method of inhibiting gene expression wherein a conjugate is transported into a cell where it can react with a macromolecule (e.g., mRNA and double-stranded DNA) to form a complex, and thus inhibit gene expression. Thus, these conjugates can be used as oligonucleotide drugs useful in treating diseases caused by the expression of specific genes.
2. Description of the Prior Art
Recent studies have indicated that oligonucleotides have potential therapeutic value. Oligonucleotides may act as antisense inhibitors of gene expression in a number of ways. Single-stranded oligodeoxynucleotides may arrest translation by forming heteroduplexes with mRNA, or may arrest transcription by forming triple-strand helices with duplex DNA. Catalytic ribonucleotides (ribozymes) may inhibit gene expression by cleaving complementary mRNAs. Furthermore, double-stranded oligonucleotides may bind to protein sites that recognize specific sequences of bases, thereby inhibiting polymerases. (Ghosh et al., Progress in Nucleic Acid Research and Molecular Biology, 42:79-126, 1992).
Two factors may limit the use of oligonucleotides as therapeutic agents. First, unmodified oligonucleotides are particularly susceptible to degradation by ubiquitous nucleases. Many of these nucleases exhibit 3xe2x80x2 to 5xe2x80x2 exonuclease activity and require a hydroxyl group at the 3xe2x80x2-end of the DNA molecule for activity. Second, the therapeutic action of oligonucleotides is limited by their low ability to enter the target cell.
Many approaches have been taken to limit the nuclease susceptibility of oligonucleotides. One approach has been to modify the phosphorous atom of the phosphodiester linkage to produce phosphorothioates, methylphosphorates, and phosphoramidates. (Uhlamann et al., Chem. Rev., 90:543-584,1990; Goodchild, Bioconjugate Chem., 1:165-186, 1990). Although this approach has given significant resistance to nuclease degradation, the ability of the oligonucleotide to support RNase-mediated degradation of RNA is compromised. (Dagel et al., Antisense Res. Develop., 1:11-20, 1991). RNase H is a nuclease which degrades only RNA that is hybridized to DNA and is commonly used to gauge the ability of an oligodeoxynucleotide to form a stable duplex with RNA. However, phosphorothioate oligonucleotides incorporating 2xe2x80x2-deoxy-2xe2x80x2-fluoroadenosine, -guanosine, -uridine, and -cytidine did enhance duplex stability as measured by melting temperature (Tm) without compromising base-pair specificity. (Kawasaki et al., J. Med. Chem., 36:831-841, 1993).
Terminal phosphorothioate and methane phosphorate modifications were shown to protect an oligonucleotide from exonucleases such as snake venom phosphodiesterase and spleen phosphodiesterase. (Stein et al., Nucleic Acids Research, 16:3209-3221, 1988; Agrawal et al., Tetrahedron Lett., 27:5575-5578, 1986). Additionally, terminal methane phosphonate modifications were useful in protecting oligodeoxynucleotides from nuclease digestion in fetal calf serum. (Tidd et al., Br. J. Cancer, 60:343, 1989).
Modification of pyrimidines at their 5- and/or 6-positions resulted in enhanced nuclease stability of deoxyoligonucleotides in fetal calf serum and did not inhibit RNase H-mediated degradation of RNA. (Sanghvi et al., Nucleic Acids Research, 21:3197-3203, 1993). Also, deoxyoligonucleotides modified at the 3xe2x80x2- and/or 5xe2x80x2-terminal phosphate with phosphoroamidate linkages blocked exonucleolytic degradation in human serum, fetal calf serum, and cell media; furthermore, these terminal modifications maintained high binding affinity of the oligonucleotide for the complementary DNA sequence as shown by thermal denaturation measurements. (Shaw et al., Nucleic Acids Research, 19:747-750, 1991).
The inclusion of an 8 base-pair sequence encoding a hairpin loop on the 3xe2x80x2 end effectively stabilized a deoxyoligonucleotide against degradation by phosphodiesterase-1 and in fetal calf serum. Thermal denaturation experiments showed that the Tm of this oligonucleotide hybridized to its complementary mRNA was unaffected by the presence of the secondary structure modification. (Kahn et al., Nucleic Acids Research, 21:2957-2958, 1993).
An oligonucleotide containing internucleotide phosphorothioates and 3xe2x80x2 alkylamine exhibited in vitro stability to nucleases in fetal calf serum and in cell culture. Additionally, treatment of HeLa cells with this oligonucleotide inhibited expression of the target gene 15 days following initial oligonucleotide treatment. Confocal microscopy revealed that an oligonucleotide with both modifications exhibited greater accumulation and release from cytoplasmic vesicles compared to an oligonucleotide containing only the 3xe2x80x2 alkylamine addition. Increased intracellular accumulation and distribution of oligonucleotides are factors known to affect their biological availability. (Tam et al., Nucleic Acids Research, 22:977-986, 1994).
Although the studies described above demonstrate that specific modifications of oligonucleotides result in enhanced resistance to nucleases, these investigations have not linked oligonucleotide modifications to improved methods of cellular uptake. Unfortunately, little is known about the interaction of oligonucleotides with cell membranes.
Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989, linked a cholesteryl group to the 3xe2x80x2-terminal internucleotide phosphorus of phosphodiester and phosphorothioate oligonucleotides in order to increase oligonucleotide solubility in cell membranes. These compounds significantly inhibited replication of human immunodeficiency virus (HIV) in tissue culture.
The presence of polylysine at the 3xe2x80x2-end of oligonucleotides complementary to the HIV-1 splice donor site resulted in a significant reduction in the production of viral structural proteins and virus tissue in infected cultures. (Stevenson et al., J. Gen. Virol., 70:2673-2682, 1989). The authors postulated that the alteration in lipid solubility imparted by an attached polylysine moiety affects both the ability of an oligonucleotide to cross the cell membrane and its intracellular distribution.
Recently, gene-transfer methods have been developed that exploit natural receptor-mediated endocytosis pathways for the delivery of DNA into cells. One such class of receptors are the lectins, which specifically recognize carbohydrate residues. Ligands for cell receptors such as asialorosomucoid (Wu et al., J. Biol. Chem., 262:4429-4432, 1987), albumin-bound insulin (Huckett et al., Biochem. Pharmacol., 40:253-263, 1990), transferrin (Wagner et al., Proc. Natl. Acad. Sci. USA, 87:3410-3414, 1990), and viral proteins (Cotten et al., Methods Enzymol., 217:618-644, 1992) have been successfully used to import DNA molecules into cells. In each case, the ligand was conjugated to DNA-binding protein and incubated with DNA. Resultant ligand-coated DNA was able to bind to receptors on the cell surface and was subsequently internalized.
Drawbacks of this approach include the difficulty of controlling the protein conjugation chemistry (Plank et al., Bioconjugate Chem., 3:533-539, 1992), and limited gene delivery due to accumulation of DNA complexes in intracellular vesicles. Zatloukal et al., Ann. N.Y. Acad. Sci., 660:136-153, 1992). Cotten et al., Proc. Natl. Acad. Sci. USA, 87:4033-4037,1990, have attempted to overcome these problems by replacing large protein ligands with smaller compounds that mimic ligand-binding to a receptor. These researchers constructed an artificial ligand for the asialoglycoprotein which contained four terminal galactose residues on a branched carrier peptide. This synthetic ligand was conjugated to polylysine-bound DNA and delivered into cultured hepatocytes.
Bonfils et al., Nucleic Acids Research, 20:4621-4629, 1992, covalently linked an oligonucleotide substituted at the 3xe2x80x2-end by a thiol group to 6-phosphomannosylated bovine serum albumin via a disulfide bridge. The conjugated oligonucleotide was stable in fetal bovine serum. The cellular uptake of the conjugation was mediated by mannose-6-phosphate membrane lectins.
Thus, the prior art reveals the difficulties researchers have encountered in developing efficient methods of delivery of oligonucleotides into cells in order to inhibit expression of specific genes.
The present invention provides an essential step in oligonucleotide therapy: the efficient delivery of oligonucleotides into cells. In this invention, an oligonucleotide is conjugated to a carbohydrate. The preferred on conjugates os the invention include (and more preferably consist essentially of) a single oligonucleotide compound conjugated to a carbohydrate with a crosslinker, as these species are herein defined. The carbohydrate of the conjugate has two roles. It may act as a ligand which can recognize a specific receptor located at the surface of the target cell. Receptor-bound conjugate then may be transported into the cell by receptor-mediated endocytosis. Furthermore, the carbohydrate of the conjugate protects the covalently attached oligonucleotide from attack by nucleases. The present invention is unique and unknown in the prior art in that no one has conjugated a nucleic acid molecule directly to a carbohydrate molecule in order to facilitate transport across cell membranes or to protect an oligonucleotide from degradation by nucleases.
In the most preferred embodiment, the oligonucleotide-carbohydrate conjugate is formed by covalently attaching a sucrose molecule to the 3xe2x80x2-end hydroxyl group of either a phosphodiester or phosphorothioate oligodeoxynucleotide via the crosslinker 1,5-bis(succinimidooxycarbonyloxy)-pentane. The oligonucleotide has a length of at least about 2 nucleotides, ideally from about 2 to 50 nucleotides. The conjugate is synthesized in reactions catalyzed by Bacillus protease type VIII (subtilisin); these reactions are carried out in anhydrous pyridine at from about 35 to 60xc2x0 C., ideally from about 40 to 50xc2x0 C., for 24 hours in a single reaction mixture with agitation of reactants. The resultant sucrose-conjugated oligonucleotides are resistant to degradation by Exonuclease-1 and by endogenous nucleases present in fetal calf serum. Furthermore, attachment of sucrose does not destroy the capacity of the oligonucleotides to form stable duplexes with RNA, since such duplexes are able to support RNase H-mediated degradation of the RNA strand.
However, the oligonucleotide, carbohydrate, crosslinker, and enzyme used in the synthesis of the conjugate are not limited to oligodeoxynucleotides, sucrose, 1,5-bis(succinimidooxycarbonyloxy)pentane, and subtilisin, respectively. Suitable oligonucleotides also include oligoribonucleotides, both catalytic (ribozymes) and uncatalytic. Selection of a carbohydrate depends upon the receptor utilized in cellular uptake of the oligonucleotide; suitable carbohydrates include sucrose, N-acetyllactosamine, galactose, glucose, mannose, Sialyl Lewisx and derivatives and fragments thereof, and carbohydrate moieties of biofunctional glycoproteins and glycopeptides having amino acid residue lengths of up 10 amino acid residues. The most preferred carbohydrates have high cell receptor binding capacities, such as sucrose, mannose and galactose. Suitable crosslinkers include the following compounds: 
wherein each R moiety is a succinimidyl, imidazolyl, benzoimidazolyl, triazolyl, or maleimidyl moiety, and each n equals from 1 to 50. Suitable enzymes include Bacillus protease bioenzyme 240, Bacillus protease B-500, alkaline protease, aminoacylase, and lipase. The invention also comprehends attachment of the carbohydrate to the oligonucleotide without the use of a crosslinker or enzyme. Those of ordinary skill in the art recognize that the conjugate can be formed in a nonenzymatic chemical reaction.
As used herein, xe2x80x9ccarbohydratexe2x80x9d refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched or cyclic) with an oxygen atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched or cyclic), with an oxygen atom bonded to each carbon atom. Generally, the preferred carbohydrates useful in the invention are of relatively low molecular weight in order to minimize the changes of the final conjugant being immunogenic in vivo; the selected carbohydrate per se or carbohydrate moiety-containing compound should therefore preferably have a molecular weight of up to about 1,500 and more preferably up to about 500. Representative carbohydrates include the sugars (mono-, di-, tri-, and oligosaccharides containing from about 4-9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C6 and above (preferably C6-C8) sugars such as glucose, fructose, mannose, galactose and sedoheptulose; di- and trisaccharides would include sugars having two or three monosaccharide units (preferably C5-C8) such as sucrose, cellobiose, maltose, lactose, and raffinose.
On the other hand, an xe2x80x9coligonucleotidexe2x80x9d is a compound made up of recurring nucleotide units, each consisting of a nitrogenous base, a 5-carbon sugar moiety (ribose in RNA and deoxyribose in DNA). An oligonucleotide is not a xe2x80x9ccarbohydratexe2x80x9d in the context of the present invention, which requires one or more C6 or greater monosaccharide units therein. In addition, oligonucleotides are characterized by specific recurrent nitrogenous basis (adenine, guanine, cytosine, uracil or thymine referred to as A, G, C, U and T respectively), and phosphodiester linkages. Preferably, the carbohydrates of the invention are essentially or completely free of both the characterizing A, G, C, U and T nitrogeneous bases and/or phosphodiester conjugation linkages between the carbohydrate and oligonucleotide.
The preferred oligonucleotide-carbohydrate conjugates of the invention should be non-proteinaceous, i.e., free of protein or peptide-like moieties having over 10 amino acid residues. Neither the oligonucleotide, the carbohydrate nor the crosslinker should therefore contain an amino acid residue moiety exceeding 10 amino acid residues in length.
The preferred class of crosslinkers includes non-naturally occurring, non-proteinaceous synthetic compounds having a molecular weight of up to 20,000 (preferably up to 10,000) and which are electrically neutral. The crosslinkers should therefore not be in the form of large proteinaceous compounds of high molecular weight and electrical charge. In the most preferred form, the molecular weight of the crosslinker should be less than, and certainly no more than, that of the oligonucleotide. In the more general case, the molecular weight of the cross linker should not be greater than two times the molecular weight of the oligonucleotide.
An oligonucleotide-carbohydrate conjugate, once transported into the target cell, may inhibit gene expression by binding to a cellular macromolecule (e.g., mRNA and double-stranded DNA) to form a complex. Thus, the oligonucleotide may hybridize to complementary mRNA, resulting in either translation arrest or degradation of the mRNA by RNase H. If the oligonucleotide is a ribozyme, it may cleave the mRNA. Alternately, the oligonucleotide may hybridize to double-stranded DNA, resulting in transcription arrest. Thus, the conjugates of the present invention may serve as therapeutic agents useful in treating diseases resulting from expression of specific genes.